Preferentially substituted calcium channel blockers

ABSTRACT

Compounds of the formula                    
     and their salts, 
     wherein Cy represents cyclohexyl; 
     Y is CH═CHΦ, CHΦ 2 , Φ or Cy, 
     X is trivalent straight-chain alkylene (3-10C) or trivalent straight-chain 1-alkenylene (3-10C) optionally substituted by oxo at the C adjacent N when n is 0 and Y is Φ 2 CH; and is otherwise trivalent straight-chain alkylene (5-10C) or trivalent straight-chain 1-alkenylene (5-10C) optionally substituted by oxo at the C adjacent N; 
     Z is N, NCO, CHNCOR 1  or CHNR 1 , wherein R 1  is alkyl (1-6C); and 
     n is 0-5; 
     wherein each Φ and Cy independently may optionally be substituted by alkyl (1-6C) or by halo, CF 3 , OCF 3 , NO 2 , NR 2 , OR, SR, COR, COOR, CONR 2 , NROCR or OOCR where R is H or alkyl (1-4C), or two substituents may form a 5-7 membered ring 
     with the proviso that the compounds of formula (1) contain at least one aromatic moiety, 
     are useful as calcium channel blockers.

This application is a continuation of U.S. Ser. No. 09/476,927 filedDec. 30, 1999, now U.S. Pat. No. 6,387,897, which is acontinuation-in-part of U.S. Ser. No. 09/401,699, filed Sep. 23, 1999now U.S. Pat. No. 6,294,533, which is a continuation-in-part of U.S.Ser. No. 09/107,037 filed Jun. 30, 1998 and now U.S. Pat. No. 6,011,035.The contents of all applications are incorporated herein by reference.

TECHNICAL FIELD

The invention relates to compounds useful in treating conditionsassociated with calcium channel function. More specifically, theinvention concerns compounds containing substituted or unsubstitutedderivatives of 6-membered heterocyclic moieties that are useful intreatment of conditions such as stroke and pain.

BACKGROUND ART

Native calcium channels have been classified by theirelectrophysiological and pharmacological properties as T, L, N, P and Qtypes (for reviews see McCleskey, E. W. et al. Curr Topics Membr (1991)39:295-326, and Dunlap, K. et al. Trends Neurosci (1995) 18:89-98).T-type (or low voltage-activated) channels describe a broad class ofmolecules that transiently activate at negative potentials and arehighly sensitive to changes in resting potential. The L, N, P and Q-typechannels activate at more positive potentials (high voltage activated)and display diverse kinetics and voltage-dependent properties. There issome overlap in biophysical properties of the high voltage-activatedchannels, consequently pharmacological profiles are useful to furtherdistinguish them. L-type channels are sensitive to dihydropyridineagonists and antagonists, N-type channels are blocked by the Conusgeographus peptide toxin, ω-conotoxin GVIA, and P-type channels areblocked by the peptide ω-agatoxin IVA from the venom of the funnel webspider, Agelenopsis aperta. A fourth type of high voltage-activatedcalcium channel (Q-type) has been described, although whether the Q- andP-type channels are distinct molecular entities is controversial(Sather, W. A. et al. Neuron (1995) 11:291-303; Stea, A. et al. ProcNatl Acad Sci USA (1994) 91:10576-10580; Bourinet, E. et al. NatureNeuroscience (1999) 2:407-415). Several types of calcium conductances donot fall neatly into any of the above categories and there isvariability of properties even within a category suggesting thatadditional calcium channels subtypes remain to be classified.

Biochemical analyses show that neuronal high voltage activated calciumchannels are heterooligomeric complexes consisting of three distinctsubunits (α₁, α₂δ and β) (reviewed by De Waard, M. et al. Ion Channels(1997) vol. 4, Narahashi, T. ed. Plenum Press, NY). The α₁ subunit isthe major pore-forming subunit and contains the voltage sensor andbinding sites for calcium channel antagonists. The mainly extracellularα₂ is disulfide-linked to the transmembrane δ subunit and both arederived from the same gene and are proteolytically cleaved in vivo. Theβ subunit is a nonglycosylated, hydrophilic protein with a high affinityof binding to a cytoplasmic region of the α₁ subunit. A fourth subunit,γ, is unique to L-type calcium channels expressed in skeletal muscleT-tubules. The isolation and characterization of γ-subunit-encodingcDNAs is described in U.S. Pat. No. 5,386,025 which is incorporatedherein by reference.

Recently, each of these α₁ subtypes has been cloned and expressed, thuspermitting more extensive pharmacological studies. These channels havebeen designated α_(1A)-α_(1I) and α_(1S) and correlated with thesubtypes set forth above. α_(1A) channels are of the P/Q type; α_(1B)represents N; α_(1C), α′_(1D), α_(1F) and α_(1S) represent L; α_(1E)represents a novel type of calcium conductance, and α_(1G)-α_(1I)represent members of the T-type family, reviewed in Stea, A. et al. inHandbook of Receptors and Channels (1994), North, R. A. ed. CRC Press;Perez-Reyes, et al. Nature (1998) 391:896-900; Cribbs, L. L. et al.Circulation Research (1998) 83:103-109; Lee, J. H. et al. Journal ofNeuroscience (1999) 19:1912-1921.

Further details concerning the function of N-type channels, which aremainly localized to neurons, have been disclosed, for example, in U.S.Pat. No. 5,623,051, the disclosure of which is incorporated herein byreference. As described, N-type channels possess a site for bindingsyntaxin, a protein anchored in the presynaptic membrane. Blocking thisinteraction also blocks the presynaptic response to calcium influx.Thus, compounds that block the interaction between syntaxin and thisbinding site would be useful in neural protection and analgesia. Suchcompounds have the added advantage of enhanced specificity forpresynaptic calcium channel effects.

U.S. Pat. No. 5,646,149 describes calcium channel antagonists of theformula A-Y-B wherein B contains a piperazine or piperidine ringdirectly linked to Y. An essential component of these molecules isrepresented by A, which must be an antioxidant; the piperazine orpiperidine itself is said to be important. The exemplified compoundscontain a benzhydril substituent, based on known calcium channelblockers (see below). U.S. Pat. No. 5,703,071 discloses compounds saidto be useful in treating ischemic diseases. A mandatory portion of themolecule is a tropolone residue; among the substituents permitted arepiperazine derivatives, including their benzhydril derivatives. U.S.Pat. No. 5,428,038 discloses compounds which are said to exert a neuralprotective and antiallergic effect. These compounds are coumarinderivatives which may include derivatives of piperazine and othersix-membered heterocycles. A permitted substituent on the heterocycle isdiphenylhydroxymethyl. Thus, approaches in the art for variousindications which may involve calcium channel blocking activity haveemployed compounds which incidentally contain piperidine or piperazinemoieties substituted with benzhydril but mandate additional substituentsto maintain functionality.

Certain compounds containing both benzhydril moieties and piperidine orpiperazine are known to be calcium channel antagonists and neurolepticdrugs. For example, Gould, R. J. et al. Proc Natl Acad Sci USA (1983)80:5122-5125 describes antischizophrenic neuroleptic drugs such aslidoflazine, fluspirilene, pimozide, clopimozide, and penfluridol. Ithas also been shown that fluspirilene binds to sites on L-type calciumchannels (King, V. K. et al. J Biol Chem (1989) 264:5633-5641) as wellas blocking N-type calcium current (Grantham, C. J. et al. Brit JPharmacol (1944) 111:483-488). In addition, Lomerizine, as developed byKanebo K K, is a known calcium channel blocker; Lomerizine is, however,not specific for N-type channels. A review of publications concerningLomerizine is found in Dooley, D., Current Opinion in CPNSInvestigational Drugs (1999) 1:116-125.

The present invention is based on the recognition that the combinationof a six-membered heterocyclic ring containing at least one nitrogensaid nitrogen coupled through a linker to a benzhydril moiety results ineffective calcium channel blocking activity. In some cases enhancedspecificity for N-type channels, or decreased specificity for L-typechannels is shown. The compounds are useful for treating stroke and painand other calcium channel-associated disorders, as further describedbelow. By focusing on these moieties, compounds useful in treatingindications associated with calcium channel activity are prepared.

DISCLOSURE OF THE INVENTION

The invention relates to compounds useful in treating conditions such asstroke, head trauma, migraine, chronic, neuropathic and acute pain,epilepsy, hypertension, cardiac arrhythmias, and other indicationsassociated with calcium metabolism, including synaptic calciumchannel-mediated functions. The compounds of the invention arebenzhydril or partly saturated benzhydril derivatives of piperidine orpiperazine with substituents which enhance the calcium channel blockingactivity of the compounds. Thus, in one aspect, the invention isdirected to therapeutic methods that employ compounds of the formula

wherein Cy represents cyclohexyl;

Y is CH═CHΦ, CHΦ₂, Φ or Cy,

X is trivalent straight-chain alkylene (3-10C) or trivalentstraight-chain 1-alkenylene (3-10C) optionally substituted by oxo at theC adjacent N when n is 0 and Y is Φ₂CH; and is otherwise trivalentstraight-chain alkylene (5-10C) or trivalent straight-chain 1-alkenylene(5-10C) optionally substituted by oxo at the C adjacent N;

Z is N, NCO, CHNCOR¹ or CHNR¹, wherein R¹ is alkyl (1-6C); and

n is 0-5;

wherein each Φ and Cy independently may optionally be substituted byalkyl (1-6C) or by halo, CF₃, OCF₃, NO₂, NR₂, OR, SR, COR, COOR, CONR₂,NROCR or OOCR where R is H or alkyl (1-4C), or two substituents may forma 5-7 membered ring

with the proviso that the compounds of formula (1) contain at least onearomatic moiety.

The invention is directed to methods to antagonize calcium channelactivity using the compounds of formula (1) and thus to treat associatedconditions. It will be noted that the conditions may be associated withabnormal calcium channel activity, or the subject may have normalcalcium channel function which nevertheless results in an undesirablephysical or metabolic state. In another aspect, the invention isdirected to pharmaceutical compositions containing these compounds.

The invention is also directed to combinatorial libraries containing thecompounds of formula (1) and to methods to screen these libraries formembers containing particularly potent calcium channel blocking activityor for members that antagonize one type of such channels specifically.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B shows a comparison of certain preferred embodiments ofthe compounds of the invention to the known compound Lomerizine.

FIG. 2 is a graphic representation of the specificity of the compoundsshown in FIG. 1 with respect to N-type, L-type and P/Q-type channels.

FIG. 3 is a graphic representation of the data shown in FIG. 2 based onIC₅₀ values calculated from the data shown in FIG. 2.

MODES OF CARRYING OUT THE INVENTION

The compounds of formula (1), useful in the methods of the invention,exert their desirable effects through their ability to antagonize theactivity of calcium channels. This makes them useful for treatment ofcertain conditions. Among such conditions are stroke, epilepsy, headtrauma, migraine and chronic, neuropathic and acute pain. Calcium fluxis also implicated in other neurological disorders such asschizophrenia, anxiety, depression, other psychoses, and certaindegenerative disorders. Other treatable conditions includecardiovascular conditions such as hypertension and cardiac arrhythmias.

While the compounds of formula (1) generally have this activity, theavailability of a multiplicity of calcium channel blockers permits anuanced selection of compounds for particular disorders. Thus, theavailability of this class of compounds provides not only a genus ofgeneral utility in indications that are affected by excessive calciumchannel activity, but also provides a large number of compounds whichcan be mined and manipulated for specific interaction with particularforms of calcium channels. The availability of recombinantly producedcalcium channels of the α_(1A)-α_(1I) and α_(1S) types set forth above,facilitates this selection process. Dubel, S. J. et al. Proc Natl AcadSci USA (1992) 89:5058-5062; Fujita, Y. et al. Neuron (1993) 10:585-598;Mikami, A. et al. Nature (1989) 340:230-233; Mori, Y. et al. Nature(1991) 350:398-402; Snutch, T. P. et al. Neuron (1991) 7:45-57; Soong,T. W. et al. Science (1993) 260:1133-1136; Tomlinson, W. J. et al.Neuropharmacology (1993) 32:1117-1126; Williams, M. E. et al. Neuron(1992) 8:71-84; Williams, M. E. et al. Science (1992) 257:389-395;Perez-Reyes, et al. Nature (1998) 391:896-900; Cribbs, L. L. et al.Circulation Research (1998) 83:103-109; Lee, J. H. et al. Journal ofNeuroscience (1999) 19:1912-1921.

Thus, while it is known that calcium channel activity is involved in amultiplicity of disorders, the types of channels associated withparticular conditions is the subject of ongoing data collection. Forexample, the association of N-type channels in conditions associatedwith neural transmission would indicate that compounds of the inventionwhich target N-type receptors are most useful in these conditions. Manyof the members of the genus of compounds of formula (1) exhibit highaffinity for N-type channels; other members of the genus maypreferentially target other channels.

There are two distinguishable types of calcium channel inhibition. Thefirst, designated “open channel blockage,” is conveniently demonstratedwhen displayed calcium channels are maintained at an artificiallynegative resting potential of about −100 mV (as distinguished from thetypical endogenous resting maintained potential of about −70 mV). Whenthe displayed channels are abruptly depolarized under these conditions,calcium ions are caused to flow through the channel and exhibit a peakcurrent flow which then decays. Open channel blocking inhibitorsdiminish the current exhibited at the peak flow and can also acceleratethe rate of current decay.

This type of inhibition is distinguished from a second type of block,referred to herein as “inactivation inhibition.” When maintained at lessnegative resting potentials, such as the physiologically importantpotential of −70 mV, a certain percentage of the channels may undergoconformational change, rendering them incapable of being activated—i.e.,opened—by the abrupt depolarization. Thus, the peak current due tocalcium ion flow will be diminished not because the open channel isblocked, but because some of the channels are unavailable for opening(inactivated). “Inactivation” type inhibitors increase the percentage ofreceptors that are in an inactived state.

Synthesis

The compounds of the invention may be synthesized using conventionalmethods. Illustrative of such methods are Schemes 1 and 2:

Alternatively, a carboxylic acid containing the benzhydril (or ΦCyCH orCy₂CH) moiety can first be synthesized and then reacted with thepiperazine (or piperidine) moiety and subsequently reduced. Tosynthesize the desired acid, an ω-bromo carboxylic acid is refluxedwith, in the case of benzhydril, triphenylphosphine in the presence ofmethyl nitrile and then treated with lithium hexamethyldisilazide in asolvent such as THF. The resulting unsaturated carboxylic acidcontaining the two phenyl substituents is then reduced as shown inScheme 1 with hydrogen on a palladium catalyst and then reacted withderivatized piperazine (or piperidine) to form the amide. The amide canthen be reduced as shown above.

Preferred Embodiments

The compounds of formula (1) are defined as shown in terms of theembodiments of their various substituents:

Particularly preferred embodiments of the compounds of formula (1) arethose wherein X is coupled to two phenyl groups. Less preferred areinstances where X is coupled to one phenyl and one cyclohexyl. Leastpreferred are those instances wherein X is coupled to two cyclohexylgroups.

As defined above, X may be a trivalent straight-chain alkylene of 5-10Coptionally substituted with oxo at the position adjacent the piperidineor piperazine ring nitrogen. Preferably, the alkylene chain is 5-8C,more preferably 5-7C, and even more preferably 5-6C. Substitution withoxo is preferred only when the length of the alkylene chain is 6-10C. Inaddition, X may be a straight-chain 1-alkenylene (5-10C) wherein theπ-bond is in the position distal to the piperidine or pyrimidine ringnitrogen. Under these circumstances, the two cyclic moieties areaccommodated by the alkenylene chain by virtue of the alkenylene chainas a vinyl substituent to each cyclic moiety. In addition, when n is 0and Y is Φ₂CH, the embodiment of X described above may also be shorterand may contain 3-10C.

Preferred embodiments of Z are N, NCO and CHNR¹ where R¹ is preferably Hbut may also be alkyl (1-6C), preferably 1-4C, more preferably 1-2C, andeven more preferably methyl (or H).

Preferred embodiments for n are 0-4, more preferably 1-2.

Any of the phenyl or cyclohexyl moieties contained in the compounds offormula (1) may be substituted, as noted above. Preferred substituentsinclude halo, especially fluoro, NO₂, alkyl (1-6C), preferably methyl,OR, preferably methoxy, NR₂, preferably dimethylamino, diethylamino,methylamino or ethylamino, acetamido, CF₃, OCF₃ and the like. Twosubstituted positions may also form a ring. Preferably, where the cyclicmoieties coupled to X are both phenyl, the phenyl groups are identicallysubstituted. Where one such moiety is phenyl and the other iscyclohexyl, the presence of a substituent on the phenyl moiety and anunsubstituted cyclohexyl moiety are preferred. It is believed thathalogenation of the compounds of the invention is helpful in modulatingthe in vivo half-life, and it may be particularly advantageous toinclude halogen substituents, such as fluoro substitutions on any phenylmoieties.

Particularly preferred are compounds MC-34D, JM-G-10, 39-1-B4, and39-45-3 shown in FIG. 1 and variously substituted forms thereof.

Thus, also preferred are forms of these enumerated compounds whichcontain different substituents on the phenyl or cyclohexyl moieties fromthose shown. Thus, also preferred are compounds having the generalformula of MC-34D wherein the two phenyl moieties attached to X containfluoro in the para position. Alternative substitutions are as shownbelow where Φ1 and Φ2 indicate the 2 phenyl groups attached to X (thenumbers being arbitrarily chosen as these phenyl groups are equivalent)and Φ3 represents phenyl group contained in Y. In addition, alsopreferred are embodiments as set forth herein where Z of MC-34D is NCOor wherein X is —CH(CH₂)—₅.

Φ1 Φ2 Φ3 2,4-dimethyl 2,4, dimethyl 4-F 4-methoxy 3-chloro 4-methyl2,4,6 trimethyl 2,4,6 trimethyl — amino amino 4-F 4-F 4-F 4-F 4-F4-methoxy 4-F 4-F 3,4-OCH₂O—

Similarly, JM-G-10 with substituents on the phenyl groups and cyclohexylgroup may be employed in the methods of the invention, preferredembodiments also include those wherein X is —CH(CH₂)—₅. Suitablesubstitutions are shown below:

Φ1 Φ2 Cy 4-F 4-F — 2,4-dimethoxy 4-methyl 3,5-diethyl 3,5-diamino3;5-diamino 4-F

Alternatively substituted compounds of formula 39-1-B4 are also includedwithin the preferred embodiments of the invention. In the table below,Φ1 and Φ2 represent the two equivalent phenyl groups coupled to X and Φ3and Φ4 represent the two equivalent phenyl groups included in Y. Formsof 39-1-B4 wherein the carbonyl group in X is reduced to methylene arealso preferred, including those with the substituents shown below.

Φ1 Φ2 Φ3 Φ4 4-F 4-F — — 3,4-CH₂CH₂CH₂— 3,4-CH₂CH₂CH₂— 4-F 4-F2,6-dimethoxy 3,5,diamino 3,5,diamino 2,6,dimethoxy 4-COOH 4-COOH 3,5dichloro 3,5-dichloro

Similarly, various alternative substitution patterns on compound 39-45-3may be employed. Included are those embodiments where a carbonyl ispresent adjacent the piperazine in the substituent X. Also included areanalogs where n=0. Particularly preferred are embodiments where twosubstituents on the phenyl group contained in Y form a ring,particularly a 5-membered ring. Thus, preferred substitution patternsare those set forth below where Φ1 and Φ2 represent the two equivalentphenyl groups attached to X and Φ3 represents the phenyl group containedin Y.

Φ1 Φ2 Φ3 4-F 4-F 3,4,5-trimethoxy 4-F 4-F 3,4,-CH₂CH₂—CH₂— 4-F 4-F3,4,5-tri trifluro-methyl 4-F 4-F 3,4,-OCH₂O— 2,4,6 trimethoxy — 4-F3,5-diethoxy 3,5-diethoxy 3,5-diethoxy 4-F 4-F 3,5-detrifluoromethyl 4-F4-F 5-OCF₃ 4-f 4-F 3,4 dimethyl 4-F 4-F 3-methoxy 4-F 4-F 4-F 4-F 4-F3-methyl 4-F 4-F 2-methoxy 4-F 4-F 4-acetyl

The pattern of substitution will influence the strength of calciumchannel blocking ability as well as specificity.

Where the structure permits, invention compounds may also be supplied aspharmaceutically acceptable salts. Pharmaceutically acceptable saltsinclude the acid addition salts which can be formed from inorganic acidssuch as hydrochloric, sulfuric, and phosphoric acid or from organicacids such as acetic, propionic, glutamic, glutaric, as well as acidion-exchange resins.

Libraries and Screening

The compounds of the invention can be synthesized individually usingmethods known in the art per se, or as members of a combinatoriallibrary.

Synthesis of combinatorial libraries is now commonplace in the art.Suitable descriptions of such syntheses are found, for example, inWentworth, Jr., P. et al. Current Opinion in Biol (1993) 9:109-115;Salemme, F. R. et al. Structure (1997) 5:319-324. The libraries containcompounds with various substitutents and various degrees ofunsaturation, as well as different chain lengths. The libraries, whichcontain, as few as 10, but typically several hundred members to severalthousand members, may then be screened for compounds which areparticularly effective against a specific subtype of calcium channel,i.e., the N-type channel. In addition, using standard screeningprotocols, the libraries, may be screened for compounds which blockadditional channels or receptors such as sodium channels, potassiumchannels and the like.

The libraries useful as sources of candidate compounds comprisecompounds of the formula

wherein m is 0, 1 or 2;

wherein when m is 0, Z is O, when m is 1, Z is N, and when m is 2, Z isC;

Y is H, OH, NH₂, or an organic moiety of 1-20C, optionally additionallycontaining 1-8 heteroatoms selected from the group consisting of N, P,O, S and halo;

each l¹ and l² is independently 0-5;

l³ is 0 or 1;

each of R¹, R² and R³ is independently alkyl (1-6C), aryl (6-10C) orarylalkyl (7-16C) optionally containing 1-4 heteroatoms selected fromthe group consisting of halo, N, P, O, and S or each of R¹ and R² mayindependently be halo, COOR, CONR₂, CF₃, CN or NO₂, wherein R is H orlower alkyl (1-4C) or alkyl (1-6C);

n is 0 or 1; and

X is a linker.

Methods of performing these screening functions are well known in theart. Typically, the receptor to be targeted is expressed at the surfaceof a recombinant host cell such as human embryonic kidney cells. Theability of the members of the library to bind the channel to be testedis measured, for example, by the ability of the compound in the libraryto displace a labeled binding ligand such as the ligand normallyassociated with the channel or an antibody to the channel. Moretypically, ability to antagonize the receptor is measured in thepresence of calcium ion and the ability of the compound to interferewith the signal generated is measured using standard techniques.

In more detail, one method involves the binding of radiolabeled agentsthat interact with the calcium channel and subsequent analysis ofequilibrium binding measurements including, but not limited to, onrates, off rates, K_(d) values and competitive binding by othermolecules. Another method involves the screening for the effects ofcompounds by electrophysiological assay whereby individual cells areimpaled with a microelectrode and currents through the calcium channelare recorded before and after application of the compound of interest.Another method, high-throughput spectrophotometric assay, utilizesloading of the cell lines with a fluorescent dye sensitive tointracellular calcium concentration and subsequent examination of theeffects of compounds on the ability of depolarization by potassiumchloride or other means to alter intracellular calcium levels.

As described above, a more definitive assay can be used to distinguishinhibitors of calcium flow which operate as open channel blockers, asopposed to those that operate by promoting inactivation of the channel.The methods to distinguish these types of inhibition are moreparticularly described in the examples below. In general, open-channelblockers are assessed by measuring the level of peak current whendepolarization is imposed on a background resting potential of about−100 mV in the presence and absence of the candidate compound.Successful open-channel blockers will reduce the peak current observedand may accelerate the decay of this current. Compounds that areinactivated channel blockers are generally determined by their abilityto shift the voltage dependence of inactivation towards more negativepotentials. This is also reflected in their ability to reduce peakcurrents at more depolarized holding potentials (e.g., −70 mV) and athigher frequencies of stimulation, e.g., 0.2 Hz vs. 0.03 Hz.

Utility and Administration

For use as treatment of human and animal subjects, the compounds of theinvention can be formulated as pharmaceutical or veterinarycompositions. Depending on the subject to be treated, the mode ofadministration, and the type of treatment desired—e.g., prevention,prophylaxis, therapy; the compounds are formulated in ways consonantwith these parameters. A summary of such techniques is found inRemington's Pharmaceutical Sciences, latest edition, Mack PublishingCo., Easton, Pa., incorporated herein by reference.

In general, for use in treatment, the compounds of formula (1) maybeused alone, as mixtures of two or more compounds of formula (1) or incombination with other pharmaceuticals. Depending on the mode ofadministration, the compounds will be formulated into suitablecompositions to permit facile delivery.

Formulations may be prepared in a manner suitable for systemicadministration or topical or local administration. Systemic formulationsinclude those designed for injection (e.g., intramuscular, intravenousor subcutaneous injection) or may be prepared for transdermal,transmucosal, or oral administration. The formulation will generallyinclude a diluent as well as, in some cases, adjuvants, buffers,preservatives and the like. The compounds can be administered also inliposomal compositions or as microemulsions.

For injection, formulations can be prepared in conventional forms asliquid solutions or suspensions or as solid forms suitable for solutionor suspension in liquid prior to injection or as emulsions. Suitableexcipients include, for example, water, saline, dextrose, glycerol andthe like. Such compositions may also contain amounts of nontoxicauxiliary substances such as wetting or emulsifying agents, pH bufferingagents and the like, such as, for example, sodium acetate, sorbitanmonolaurate, and so forth.

Various sustained release systems for drugs have also been devised. See,for example, U.S. Pat. No. 5,624,677.

Systemic administration may also include relatively noninvasive methodssuch as the use of suppositories, transdermal patches, transmucosaldelivery and intranasal administration. Oral administration is alsosuitable for compounds of the invention. Suitable forms include syrups,capsules, tablets, as in understood in the art.

For administration to animal or human subjects, the dosage of thecompounds of the invention is typically 0.1-15 mg/kg, preferably 0.1-1mg/kg. However, dosage levels are highly dependent on the nature of thecondition, the condition of the patient, the judgment of thepractitioner, and the frequency and mode of administration.

The following examples are intended to illustrate but not to limit theinvention.

EXAMPLE 1 Assessment of Calcium Channel Blocking Activity

Antagonist activity was measured using whole cell patch recordings onhuman embryonic kidney cells either stably or transiently expressing ratα_(1B)+α_(2b)+β_(1b) channels (N-type channels) with 5 mM barium as acharge carrier.

For transient expression, host cells, such as human embryonic kidneycells, HEK 293 (ATCC# CRL 1573) were grown in standard DMEM mediumsupplemented with 2 mM glutamine and 10% fetal bovine serum. HEK 293cells were transfected by a standard calcium-phosphate-DNAcoprecipitation method using the rat α_(1B)+β_(1b)+α₂δ N-type calciumchannel subunits in a vertebrate expression vector (for example, seeCurrent Protocols in Molecular Biology).

After an incubation period of from 24 to 72 hrs the culture medium wasremoved and replaced with external recording solution (see below). Wholecell patch clamp experiments were performed using an Axopatch 200Bamplifier (Axon Instruments, Burlingame, Calif.) linked to an IBMcompatible personal computer equipped with pCLAMP software. Borosilicateglass patch pipettes (Sutter Instrument Co., Novato, Calif.) werepolished (Microforge, Narishige, Japan) to a resistance of about 4 MΩwhen filled with cesium methanesulfonate internal solution (compositionin MM: 109 CsCH₃SO₄, 4 MgCl₂, 9 EGTA, 9 HEPES, pH 7.2). Cells werebathed in 5 mM Ba⁺⁺ (in mM: 5 BaCl₂, 1 MgCl₂, 10 HEPES, 40tetraethylammonium chloride, 10 glucose, 87.5 CsCl pH 7.2). Current datashown were elicited by a train of 100 ms test pulses at 0.066 Hz from−100 mV and/or −80 mV to various potentials (min. −20 mV, max. +30 mV).Drugs were perfused directly into the vicinity of the cells using amicroperfusion system.

Normalized dose-response curves were fit (Sigmaplot 4.0, SPSS Inc.,Chicago, Ill.) by the Hill equation to determine IC₅₀ values.Steady-state inactivation curves were plotted as the normalized testpulse amplitude following 5 s inactivating prepulses at +10 mVincrements. Inactivation curves were fit (Sigmaplot 4.0) with theBoltzman equation, I_(peak) (normalized)=1/(1+exp((V−V_(h))z/25.6)),where V and V_(h) are the conditioning and half inactivation potentials,respectively, and z is the slope factor.

EXAMPLE 2 Synthesis of Illustrative Compounds of Formula (1)

A. Synthesis of 6,6-Diphenyl Hexanoic Acid.

6-Bromohexanoic acid (7.08 g, 36.3 mmole) and triphenylphosphine (10 g,38.2 mmole) were mixed in dry CH₃CN (40 ml), heated to reflux overnightand allowed to cool to RT. The solution was concentrated under reducedpressure to give a viscous gel. Approximately 75 ml of THF was added tothe reaction mixture and the walls of the flask were scratched with aspatula to start crystallization. The resulting solid was filtered undervacuum, washed with THF and dried under reduced pressure and usedwithout further purification.

This product (1.5 g) was suspended in dry THF (10 ml) and the flaskpurged with N₂ and cooled to −78° C. To the stirred reaction was addedlithium hexamethyldisilazide (LiHMDS) (10 ml, 1 M in THF). The yellowsolution was stirred at −78° C. for 1 h over which time the reactiondarkened slightly. The cooling bath was removed and the reaction allowedto warm to RT. The reaction was kept at RT for 1 h during which time thesolution turned a dark red color and most of the solids went intosolution. Benzophenone (0.54 g in 3 ml THF) was added to the reactionand allowed to react overnight. The yellow solution was concentratedunder reduced pressure to give a yellow solid. The resulting solid waspartitioned between ether and 10% HCl. The organic layer was washed withwater (2×) and extracted with 10% NaOH (3×). The combined aqueous basefraction was acidified with conc. HCl to a pH of 4. The water layer wasextracted with ether (3×) and the combined organic fractions dried overNa₂SO₄.

The ether was evaporated to dryness under reduced pressure to give acolorless oil which crystallized on standing to give a waxy solid,6,6-diphenyl hex-5-enoic acid, which was dissolved in 30 ml MeOH andmixed with 5% Pd—C and placed in a Parr hydrogenator. The reactionvessel was purged with hydrogen and pressurized to 60 PSIG and reactedat RT for 4 h. The reaction mixture was sampled and analyzed by TLC. Ifthe TLC when stained with KMnO₄ showed a positive test for alkenes thereaction mixture was resubjected to the reaction conditions. Thesolution was then filtered through a plug of celite and the methanolfiltrate containing 6,6-diphenyl hexanoic acid was concentrated undervacuum.

B. Reaction with Substituted Piperazine.

6,6-Diphenylhexanoic acid (0.4 mmoles) was mixed with the desiredsubstituted piperazine (0.35 mmoles) in dry THF (7 ml). EDC (0.5 mmoles)and DMAP (cat) were added and the mixture heated to 40° C. with shakingovernight. The reaction was diluted with ethyl acetate and washed withwater (4×) and 10% NaOH (3×) and dried over sodium sulfate andevaporated to dryness. The resulting residue was purified by columnchromatography (silica gel, 1:1 hexane:EtOAc), and the products werecharacterized by HPLC-MS.

Piperazines used in the foregoing procedure include phenylpiperazine,benzylpiperazine, benzhydrilpiperazine, and piperazine substituted atthe 1-position with Φ—CH═CH₂—.

The resulting compounds contain a carbonyl adjacent to the ring nitrogenof piperazine. These compounds are of formula (1) and exhibit calciumchannel blocking activity.

C. Reduction of CO.

The compounds prepared in paragraph B were dissolved in dry THF (5 ml)and reacted with LiAlH₄ (1 M in THF) and allowed to react for 6 h. Thereactions were quenched with EtOAc (15 ml) and extracted with water (5×)10% NaOH (10×), brine (1×), dried over sodium sulfate and concentratedunder reduced pressure. Most of the products at this stage were >80%pure. Those <80% were purified for running a short column (silica gel,1:1 hex:EtOAc).

EXAMPLE 3 Preparation of Compounds of Formula (1) fromBenzhydrilpiperazine Derivatives

N-(Diphenylmethyl)piperazine (0.5 mmole) was dissolved in dry THF (10ml). To each reaction flask was added powdered K₂CO₃ and acid chlorideof the formula Y—CO—Cl (0.7 mmole). The reaction was stirred at RT for 2h and quenched with 105 NaOH (10 ml) and extracted with EtOAc (10 ml).The organic layer was washed with 10% NaOH (4×) and dried over sodiumsulfate, concentrated, and purified by column chromatography (silicagel, 1:1 hex:EtOAc) to give the desired amide. Acyl halides used in thisprocedure included cyclohexyl COCl, ΦCOCl and ΦCH═CHCOCl.

To reduce the resulting amide, the above product was dissolved in dryTHF (5 ml) and reacted with LiAlH₄ (1 M in THF) and allowed to react for6 h. The reaction was quenched with EtOAc (15 ml) and extracted withwater (5×) 10% NaOH (10×), brine (1×), dried over sodium sulfate andconcentrated under reduced pressure. Most of the products at this stagewere >80% pure. Those <80% were purified for running a short column(silica gel, 1:1 hex:EtOAc).

EXAMPLE 4 Channel Blocking Activities of Various Invention Compounds

Using the procedure set forth in Example 1, various compounds of theinvention were tested for their ability to block N-type calciumchannels. The results are shown in the table below where IC₅₀ is givenin μM (micromolar).

X coupled to X Z n Y IC₅₀ φ, φ CHCH₂ CHNH 1 φ ±3 φ, φ CH(CH₂)₂ CHNH 1 φ2 φ, φ CHCH₂ CHNH 1 Cy 3-4 φ, φ CH(CH₂)₂ CHNH 1 Cy 2-3 φ, φ CH(CH₂)₄COCHNH 1 Cy 0.75 φ, φ C═CH(CH₂)₂ N 1 Cy 5.2 φ, Cy CH(CH₂)₅ N 1 CH═CHφ 5.9φ, Cy CH(CH₂)₄CO N 1 CH═CHφ 3.9 φ, Cy CH(CH₂)₅CO N 1 CH═CHφ 3.2 φ, CyCH(CH₂)₅CO N 0 CHφ₂ 10.2 φ, Cy CH(CH₂)₅CO N 1 CH═CHφ 12.2 φ, Cy CH(CH₂)₆N 1 Cy 7.2 φ, Cy CHCH₂ N 1 Cy 20.2 φ, Cy CHCO N 1 φ 14.2 φ, Cy CHCO N 1CH═CHφ 5.9 φ, Cy CHCH₂ N 1 φ ±5 φ, Cy CH(CH₂)₅ N 2 φ 3.1 φ, Cy CHCO N 1CH═CHφ ±5 φ, Cy CHCH₂ N 1 CH═CHφ 10.6 φ, Cy CHCH₂ N 1 CH═CHφ ±5 φ, CyCHCO N 1 φ 20 φ, Cy CHCO N 1 CHφ₂ 35 φ, Cy CHCH₂ N 1 CHφ₂ 20

EXAMPLE 5 Additional Methods

The methods of Examples 1 and 2 were followed with slight modificationsas will be apparent from the description below.

A. Transformation of HEK Cells:

N-type calcium channel blocking activity was assayed in human embryonickidney cells, HEK 293, stably transfected with the rat brain N-typecalcium channel subunits (α_(1B)+α_(2δ)+β_(1b) cDNA subunits).Alternatively, N-type calcium channels (α_(1B)+α_(2δ)+β_(1b) cDNAsubunits), L-type channels (α_(1C)+α_(2δ)+β_(1b) cDNA subunits) andP/Q-type channels (α_(1A)+α_(2δ)+β_(1b) cDNA subunits) were transientlyexpressed in HEK 293 cells. Briefly, cells were cultured in Dulbecco'smodified eagle medium (DMEM) supplemented with 10% fetal bovine serum,200 U/ml penicillin and 0.2 mg/ml streptomycin at 37° C. with 5% CO₂. At85% confluency cells were split with 0.25% trypsin/1 mM EDTA and platedat 10% confluency on glass coverslips. At 12 hours the medium wasreplaced and the cells transiently transfected using a standard calciumphosphate protocol and the appropriate calcium channel cDNAs. Fresh DMEMwas supplied and the cells transferred to 28° C./5% CO₂. Cells wereincubated for 1 to 2 days to whole cell recording.

B. Measurement of Inhibition:

Whole cell patch clamp experiments were performed using an Axopatch 200Bamplifier (Axon Instruments, Burlingame, Calif.) linked to a personalcomputer equipped with pCLAMP software. The external and internalrecording solutions contained, respectively, 5 mM BaCl₂, 1 mM MgCl₂, 10mM HEPES, 40 mM TEACl, 10 mM glucose, 87.5 mM CsCl (pH 7.2) and 108 mMCsMS, 4 mM MgCl₂, 9 mM EGTA, 9 mM HEPES (pH 7.2). Currents weretypically elicited from a holding potential of −80 mV to +10 mV usingClampex software (Axon Instruments). Typically, currents were firstelicited with low frequency stimulation (0.03 Hz) and allowed tostabilize prior to application of the compounds. The compounds were thenapplied during the low frequency pulse trains for two to three minutesto assess tonic block, and subsequently the pulse frequency wasincreased to 0.2 Hz to assess frequency dependent block. Data wereanalyzed using Clampfit (Axon Instruments) and SigmaPlot 4.0 (JandelScientific).

Specific data obtained for N-type channels are shown in Table 1 below.As indicated by the data in Table 1, the most potent inhibitors athigher frequencies were MC-34D, JM-G-10, 39-1-B4 and 39-45-3 shown inFIG. 1. However, all of the compounds tested appeared to be reasonablygood blockers at this frequency.

TABLE 1 % Block Estimated IC₅₀ (100 nM) (μM) α_(1B) (N-type) 0.03 Hz 0.2Hz 0.03 Hz 0.2 Hz MC-34D 47 73 0.12 0.05 JM-G-10 38 63 0.17 0.06 39-1-B434 72 0.2  0.04 SH-123A 19 55 0.47 0.09 SH-123B  9 45 1.27 0.13 SH-13420 41 0.44 0.15 SH-136 14 44 0.63 0.14 39-45-3 50 79 0.1  0.03 39-36-1Lomerizine 24 54 0.39 0.09 39-36-2 29 60 0.32 0.07

Tables 2 and 3 show the results of similar experiments conducted withP/Q-type and L-type channels expressed in HEK 293 cells. In general, theIC₅₀ values for MC-34D, JM-G-10, 39-1-B4 and 39-45-3 were higher thanthose exhibited with respect to N-type channels.

TABLE 2 % Block Estimated IC₅₀ (100 nM) (μM) α_(1A) (P/Q-type) 0.03 Hz0.2 Hz 0.03 Hz 0.2 Hz MC-34D 31 56 0.35 0.08 JM-G-10 28 62 0.3  0.0739-1-B4 31 52 0.65 0.12 SH-123A 29 61 0.26 0.07 SH-123B 16 44 0.55 0.13SH-134 20 45 0.45 0.12 SH-136 18 42 0.52 0.15 39-45-3 27 58 0.41 0.0939-36-1 Lomerizine 25 57 0.46 0.07 39-36-2 35 52 0.31 0.11

TABLE 3 % Block Estimated IC ₅₀ (100 nM) (μM) α_(1C) (L-type) 0.03 Hz0.2 Hz 0.03 Hz 0.2 Hz MC-34D 14 24 0.63 0.39 JM-G-10 7 15 1.17 0.339-1-B4 19 29 0.5 0.3 SH-123A 21 37 0.62 0.25 SH-123B 8 14 2.06 0.74SH-134 11 21 1.6 0.83 SH-136 4 8 2.8 1.4 39-45-3 15 26 0.74 0.37 39-36-1Lomerizine 26 44 0.29 0.15 39-36-2 11 22 2.1 0.4

These data are summarized in Table 4 which show the ratio of IC₅₀ valuesfor P:N and L:N channels. As shown, with respect to specificity forL-type channels in particular, the four compounds mentioned above showmuch higher affinity for N-type and P/Q-type versus L-type channels.

TABLE 4 0.2 Hz N P/Q L P:N L:N MC-34D 0.05 0.08 0.39 1.6:1 8:1 JM-G-100.06 0.07 0.3 1.2:1 5:1 39-1-B4 0.04 0.12 0.3   3:1 8:1 SH-123A 0.090.07 0.25 0.8:1 3:1 SH-123B 0.13 0.13 0.74   1:1 6:1 SH-134 0.15 0.120.83 0.8:1 6:1 SH-136 0.14 0.15 1.4 1.1:1 10:1  39-45-3 0.03 0.09 0.37  3:1 12:1  39-36-1 Lomerizine 0.09 0.07 0.15 0.8:1 1.7:1   39-36-2 0.070.11 0.4 1.6:1 5.7:1  

These results are shown graphically in FIGS. 2 and 3.

What is claimed is:
 1. A method to identify a blocker of ion channelactivity which method comprises contacting cells which display an ionchannel of interest with a candidate compound under conditions whereinactivation of said ion channel would occur in the absence of saidcompound; and assessing the ability of said candidate compound toinhibit said activation whereby a candidate compound whose presenceresults in inhibition of activation is identified as a blocker of saidion channel, and wherein said library comprises compounds of the formula

wherein m is 0, 1 or 2; wherein when m is 0, Z is 0, when m is 1, Z isN, and when m is 2, Z is C; Y is H, OH, NH₂, or an organic moiety of1-20C, optionally additionally containing 1-8 heteroatoms selected fromthe group consisting of N, P, O, S and halo; each l¹ and l² isindependently 0-5; l³ is 0 or 1; each of R¹, R² and R³ is independentlyalkyl (1-6C), aryl (6-10C) or arylalkyl (7-16C) optionally containing1-4 heteroatoms selected from the group consisting of halo, N, P, O, andS or each of R¹ and R² may independently be halo, COOR, CONR₂, CF₃, CNor NO₂, wherein R is H or lower alkyl (1-4C) or alkyl (1-6C); n is 0 or1; and X is a linker.
 2. A method to identify a compound which is ablocker of ion channel activity which method comprises contacting a cellwhich displays said ion channel on its surface with a labeled compoundknown to bind said ion channel, said contacting in the presence andabsence of said candidate compound and assessing the ability of saidcandidate compound to inhibit the binding of said labeled ligand,whereby a candidate compound whose presence results in inhibition of thebinding of said ligand is identified as a blocker of said ion channel,and wherein said candidate compound is a member of a library whichcomprises compounds of the formula

wherein m is 0, 1 or 2; wherein when m is 0, Z is 0, when m is 1, Z isN, and when m is 2, Z is C; Y is H, OH, NH₂, or an organic moiety of1-20C, optionally additionally containing 1-8 heteroatoms selected fromthe group consisting of N, P, O, S and halo; each l¹ and l² isindependently 0-5; l³ is 0 or 1; each of R¹, R² and R³ is independentlyalkyl (1-6C), aryl (6-10C) or arylalkyl (7-16C) optionally containing1-4 heteroatoms selected from the group consisting of halo, N, P, O, andS or each of R¹ and R² may independently be halo, COOR, CONR₂, CF₃, CNor NO₂, wherein R is H or lower alkyl (1-4C) or alkyl (1-6C); n is 0 or1; and X is a linker.